I. Mullerian Inhibiting Substance
Mullerian Inhibiting Substance (MIS) is a 140,000 dalton glycoprotein responsible for the regression of the Mullerian duct in a male embryo (Jost, A., Comptes Rend. Soc. Biol., 140:463-464 (1946); Jost, A., Comptes Rend. Soc. Biol., 141:135-136 (1947); Balanchard, M. G., et al., Ped. Res., 8:968-971 (1974); Donahoe, P.K., et al., Biol. Repro., 15:329-334 (1976); Donahoe, P. K., et al., J. Ped. Surg., 12:323-330 (1977); Donahoe, P. K., et al., Biol. Repro., 16:238-243 (1977)).
Mullerian Inhibiting Substance has been found to be a glycoprotein hormone. The substance is produced by fetal and neonatal Sertoli cells of the testes. MIS has been partially purified and found to be a dimeric glycoprotein of 72,000 and 74,000 daltons (Budzik, G. P., et al., In: Lash, J. W., Saxen, L., (Eds.), Developmental Mechanisms: Normal and Abnormal. N.Y., Alan R. Liss, pp. 207-223 (1985)). The purification of MIS is described in Donahoe, P. K., et al. (U.S. Pat. No. 4,510,131).
Monoclonal antibodies to MIS have been developed and found to be useful in the purification and preparation of MIS (Shima, H., et al., Hybridoma 3:201-214 (1984); Donahoe, P. K., et al., U.S. Pat. No. 4,487,833) Necklaws, E. C., et al., Endocrinology 118:791-796 (1986). Using these monoclonal antibodies, a radioimmunoassay has been developed to detect MIS (Hayashi, M., et al., J. Histochem. Cytochem., 32:649-654 (1984)). This radioimmunoassay has detected MIS in the follicular fluid of mature bovine ovaries (Vigier, B., et al., Endocrinol., 114:1315-1320 (1984), in fluid from large and small follicles of new born ovaries (Necklaws, E., et al., Endocrinol., 2:791-796 (1986), and in incubation media of bovine granulosa cells (Vigier, B., et al., supra (1984)).
MIS has been found to be cytotoxic to human ovarian cancer cells in vitro (Donahoe, P. K., et al., Science, 205:913-915 (1979)). It has also been found to be effective against this cancer in vivo (Donahoe, P. K., et al., U.S. Pat. No. 4,510,131).
The gene for MIS has been cloned and expressed in tissue culture cells; both the DNA and amino acid sequences are known (Cate, R. L. et al., Cell 45:685-698 (1986)).
II. Mammalian Embryology
The mammalian oocyte enters the first meiotic division during fetal life, but becomes arrested in late prophase (in the dictyate or diffuse diplotene stage of meiosis) before or just after birth (Beaumont, H. M., et al., Proc. R. Soc. London (Series Biological Sciences) 155:557-579 (1962). Resumption of meiosis normally does not occur until shortly before ovulation when a surge of gonadotropins prompts the resumption of meiotic maturation (Dekel, N., et al., Proc. Natl. Acad. Sci. U.S.A., 75:4369-4373 (1978). Preovulatory resumption of meiosis is characterized by: (1) the breakdown of structures known as germinal vesicle (GV), (2) the expulsion of the first polar body and (3) progress to the metaphase of the second meiotic division (Tsafriri, A., et al., J. Repro. Fertil., 64:541-551 (1982). In the rat, the period of MIS production by the ovary coincides with the period of oocyte meiotic arrest. The physiological mechanisms responsible for keeping the oocyte in the state of meiotic arrest are unknown but are not important to an understanding of the present invention. Such mechanisms are presumed to be regulated by the MIS of the present invention. It is, however, well-known that if rodent, bovine, or porcine oocytes are cultured in vitro, and subsequently isolated from follicles, they resume meiosis spontaneously (Edwards, R. G., Nature, 196:446-450 (1962); Foote, W. D., et al., Anal. Biol. Animale Bioch. Biophys. (Paris), 9:329-(1969)).
III. Inhibitors of Meiosis
Three different types of molecules have been reported to be involved in meiosis inhibition in mammalian oocytes: (1) a low-molecular weight protein fraction of follicular fluid or granulosa cells (termed "oocyte meiosis inhibitor"), (2) steroids, and (3) cAMP and other nucleotides (Downs, S. M., et al., Proc. Natl. Acad. Sci. U.S.A., 82:454-458 (1985); McGaughey, R. W., Endocrinol., 100:39-45 (1977); Dekel, N., et al., Biol. Repro., 20:191-197 (1979); and Hubbard, C. J., et al., Biol. Repro., 26:628-632 (1982)). Oocyte meiosis inhibitor is produced by granulosa cells, and is present in the Mullerian follicular fluid. Partial purification of this fluid has revealed the oocyte meiosis inhibitor to be a small polypeptide with a molecular weight of less than 6,000. Sato, E. et al., Differentiation, 26:59-62 (1984). This protein prevents the spontaneous resumption of meiosis in cultured cumulus cell-enclosed oocytes, but not in denuded oocytes. Hillensjo, T. et al., Adv. Exper. Med. Biol., 147:175-188(1982). The protein' s inhibitory effect can be overcome by Luteinizing Hormone (LH). The inhibitory effects can also be reversed by removing the factor from the culture medium (Sato, E. et al, supra).
Thus, in summary, the background art discloses that MIS, a high molecular weight glycoprotein, is produced by mammalian testes. MIS is capable of directing the regression of the Mullerian duct in a developing male embryo. In the absence of this substance, the Mullerian duct would continue its development into the female reproductive system.
Once formed in the developing embryo, the subsequent development of mammalian oocytes becomes arrested in the late prophase stage of meiosis. This inhibition of development is believed to be the result of interactions between the female reproductive system and either a low molecular weight oocyte meiosis inhibitor, steroids, or nucleotides, such as cAMP.